In many tubular electrophoretic systems, particularly those involving capillary tubes, a bulk flow is imposed on the solutes in addition to their electrophoretic migration. Bulk flow serves several purposes, depending on the system. In systems where the solutes include both positively charged and negatively charged species, the bulk flow assures that all solute zones move in the same direction. In some systems, bulk flow is effective in increasing the speed of the analysis and minimizing the occurrence of zone broadening. Still further, bulk flow may be used to facilitate on-column sample injection and solute detection, and further to use these features to allow for operation by automated instrumentation.
One method of achieving bulk flow is through the use of an electrokinetic potential giving rise to electroendosmosis (also referred to as electroosmotic flow). Electroosmotic flow avoids the need for pumps or external devices in general. Also, by generating the driving force directly in the tube, electroosmotic flow avoids some of the problems inherent in fluid flow transmission, such as parabolic flow profiles and other effects of interfering forces such as wall shear and dead volumes.
As is well known, electroosmotic flow is the result of a surface charge developed on the inner wall of the tubing, which draws species of the opposite charge toward the wall from the bulk fluid, leaving the core region of the bulk fluid with a net charge of the same polarity as the surface charge on the wall. This net charge responds to the electric field imposed during electrophoresis to cause the bulk flow. An unfortunate property of this surface charge on the wall, however, is its tendency to attract charged solutes in the bulk fluid and thus to cause their adsorption on the wall. This continues during the electrophoretic process, gradually lessening the magnitude of the electroosmotic force and thus the bulk flow. Band broadening is frequently a result, detracting from the sensitivity and accuracy of the analysis. In addition, the adsorption of species interferes with or prevents their detection, resulting in misleading results regarding their presence in the sample. Charged impurities may in fact not be detected at all. The adsorption is gradual and in many cases irreversible, resulting in shortened useful lives of the tubing materials as well as uncertainties and inaccuracies in the analyses.
It has now been discovered that many if not all of the detrimental effects of solute adsorption are lessened and in many cases eliminated by at least partial removal of the electroosmotic force from the portion of the system where the electrophoretic separation takes place. Thus, the separation tube is separated into two regions, one where the dominating contribution is electrophoretic separation rather than any effects of surface charges, and the other where a surface charge on the wall generates most if not all of the bulk flow for the entire system.
Expressed otherwise, the invention resides in a system where one length of tubing is inert with respect to at least a portion of the solutes in the sample (i.e., the tubing material does not interact with these solutes, either by electrostatic, affinity-based, hydrophobic or other types of interaction), and another length of tubing gives rise to sufficient electrokinetic potential to drive the bulk flow through both lengths. A detector will be arranged to detect zones formed in the separation (inert) tubing without having passed through the tubing in which the electroosmotic flow is generated.
The separation may be totally inert, i.e., inert with respect to all solutes and solvents, inert only with respect to particular solute/solvent systems, or inert with respect to only a portion of the system components with selective binding of the remainder either through affinity-based or other types of interactions. The latter type of system is useful in combining electrophoretic separations with chromatographic separations.
The two lengths of tubing are joined in fluid communicating manner so that the flow generated in the electroosmotic tubing is transmitted to the separation tubing. The relative positions of the two tubing lengths with respect to the direction of flow may vary, as may the location of sample injection point and the detector, in accordance with the system parameters. These parameters may include the types of solutes to be separated and the nature of the sample in which they are contained, as well as the type of separation desired.
Further advantages and embodiments of the invention will be apparent from the following description. Expressed in a still further manner the invention resides in the use of two lengths or regions of tubing, one having a surface charge density which is substantially greater than that of the other. The predominating effect in one will thus be the electroosmotic force, while the predominating effect in the other will be electrophoretic mobility.